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Assessment of selenium status in biological material
Nuclear Instruments North-Holland
and Methods
in Physics Research
RIONil B
B75 (1993) 169-172
Beam Interactions with Materials&Atoms
Assessment of selenium status in biological material * M.C. Buoso ‘, S. Galassini a,b, M. Makarewicz ‘, F. Monti a,d, G. Moschini ‘TV,R. Ogris ‘, 0. Valkovic e and V. Valkovic Wa INFN, Laboratori Nazionali di Legnaro, Legnaro (Padova), Italy ’ Institute of Biological Chemistry, University of Verona, Italy ’ IAEA, Agency’s Laboratories, Seibersdorf; Austria d Physics Department, University of Padocla, Italy e Institut R. Boskovic, Zagreb, Croatia
Selenium is an essential trace element to man. Assessment of the selenium status, including its determination in biological materials, has been improved by the advances in analytical methodology. In this report we present the procedures used for selenium determination in different biological material by using nuclear analytical techniques, in particular PIXE, XRF and NAA. The difficulties associated with quality control of these measurements are discussed in some details. Examples of selenium determination in material are presented.
1. Introduction
There is clear, hard evidence that a geochemical environment can contribute to the maintenance of health in positive way [Il. Looking at the negative side, there is a sound evidence that a geochemical environment can be causally related to many diseases. Recently much attention has been paid to the group of elements called essential trace elements, one of them being selenium. Selenium occurs in nearly all materials of the Earth’s crust in variable concentrations rarely exceeding 0.05 ppm. Because of its variability in soil, the daily intake of selenium from food is variable and can range from 10 to 300 kg. Human activities are significantly impacting the global Se cycle, for example 1.5-2.5 times as much Se is mobilized through coal burning as by natural weather processes. LJpon combustion, coal-derived Se is preferentially partitioned to the submicrometer sized particles, which are easily transported over long distances. Selenium distribution in human tissues seems to reflect the existence of several pools for this element. Among the blood cells, platelets show a higher content of selenium than red blood cells. Selenium, when absorbed into blood, is translocated to various organs with a whole-body biological half-life of 34 d. Urine is the major route of excretion. Selenium levels in kidney, liver, spleen and heart are significantly correlated, indi* Work partially supported by the Commission pean Communities (Contract N. CI 1-0331-I(A)) Veneto (Contract N. 219/03/88).
of the Euroand Regione
0168-583X/93/$06.00
Publishers
0 1993 - El sevier Science
eating that for selenium no particular tissue (for instance the liver) is involved in selenium storage, but that selenium stores are distributed over the body. Some reports indicate a strong and significant correlation between hair selenium and liver selenium. When present in soluble forms Se is readily absorbed by plants, though differences between plant species are commonly observed. Several studies indicate that high levels of Se can inhibit cell proliferation and that the likely mechanism of action involved decreases protein synthesis. Most of these studies have demonstrated selenite to inhibit the growth and/or viability of tumor cells in culture. Recent studies have produced evidence that selenium may prevent cancer and stimulate the immune apparatus [2]. The main physiological role of this trace element is connected with its presence in various enzymes. Selenium is a constituent of enzyme glutathione peroxidase, protecting membranes from damage caused by the peroxidation of lipids. The aim of the present work is to demonstrate the use of nuclear analytical techniques in assessing the selenium status of the biological material. Although large number of selenium determination in blood, urine, tissue and different bioindicators have been performed, in this report we shall present results of measurement of Se and some other trace elements in oak tree leaves. The selection of oak free leaves as a Se exposure indicator was done because of i> the total volume of leaves being almost equal to the volume of roots and ii) the period of total leave replacement, corresponding to the period of Se integration, being three years.
B.V. All rights reserved
III. BIOMEDICAL
SAMPLES
170
MC. Buoso et al. / Selenium in biological material
2. Experimental
Leaves from oaks in two regions of Northern Italy (USL 13 and USL 141, one with increased cancer mortality, have been collected. The leaves were collected (1 kg from each tree) using surgical gloves, and were put in polyethylene bags. First of all, in order to remove dust particles, a blow of compressed air was used. Subsequently, the leaves were dried to constant weight in an oven at a temperature of about 100°C. Finally, clean, dry samples were ground in a blender, thoroughly mixed and then stored in new polyethylene bags. The analytical methods and elements determined are as follows, i) PIXE: Se (with preconcentration), K, Ca, Ti, Mn, Fe, Ni, Cu, Zn, Br, Rb, Sr, Pb; ii) XRF: Fe, Zn; iii) NAA: Se, Cr, Fe, Zn, Co. Proton induced X-ray emission spectroscopy measurements were performed with 1.8 MeV proton beam from AN 2000 Van de Graaff accelerator of the Laboratori Nazionali di Legnaro, Padova, Italy and with 3.0 MeV proton beam from EN tandem Van de Graaff accelerator of the Ruder Boskovic Institute, Zagreb, Croatia. Neutron activation analysis was done by sample irradiation with thermal neutrons in research reactor of Oesterreichische Forschungszentrum, Seibersdorf, Austria. X-ray fluorescence measurements were performed in the Laboratory for Nuclear Microanalysis, Ruder Boskovic Institute, Zagreb by using an X-ray tube excitation system with a MO anode. 2.1. PIXE measurements Samples for Se determination were prepared by the following procedures 131: 500 mg of leaf powder from each tree were placed in a 100 ml Pyrex flask with 13 ml of a mixture of 65% HNO, and 70% HCIO,, and heated to 120°C to evaporate perchloric acid 131.Then, 30% HCl was added until the HCi concentration reached 3M. After cooling, 600 pg of tellurium from a solution of KzTeO, (600 pgfml in 1N HCl) were added both as coprecipitating agent and as internal standard. Se and Te were then reduced to their elementary form by adding 4 ml of hydrazine dihydrochloride and 10 ml of HaSO,. After the selected reaction time (2 h), the precipitate was filtered using a suction Pyrex apparatus over a 0.4 km pore diameter Nucleopore. The diameter of the deposit was 10 mm. Accuracy of the measurements was verified by comparison with the Certified Standards Materials, NIST SRM 1571 Orchard Leaves (Se certified value: 0.08 +
0.01 mg/kg) and SRM 1568a Rice Flour (Se certified value: 0.4 rf:0.1 mg/kg). Samples for other trace element determinations were prepared from 1 g of fine leaf powder, without any pretreatment, as 5 mm thick pellets of 13 mm in diameter, using a stainless steel hand-press pellet maker. Samples were covered by aluminized Mylar in order to prevent charge buildup. In this case NIST SRM 1572, cytrus leaves, was used as standard reference material. The same samples were used for XRF measurements. 2.2. NAA measurements The samples were homogenized in the following way: Leaves were removed from the main rib and were grounded in a blender with a titanium knife. A fraction of the material was homogenized by the brittle fraction technique, using teflon boats cooled to the temperature of liquid nitrogen. Subsamples of about 0.2 g were weighted into quartz ampoules made from ultrapure quartz. A sufficient number of elemental standards
Table 1 Selenium concentration in Holm oak leaves in district USL 14 (w&z/kg) Sample code
NAA
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
55+ 47* 62f 74* 74* 91* 74* 14.5 + 43+ 70f 47+ 42+ 31+ 23+ 30* 24+ 47+ 41+
19 20 21 22 23 24 25 26 27
20* 215 17* 39*
PIXE 4 4 4 5 5 5 5 10 4 3 3 4 2 1 2 2 2 2 2 2 1 2
57+
64+
6
8
91% 5 1so+ 57+ 65& 70+ 62t 34*
20 5 7 7 5 7
21*
4
34+
6
34+ 22+
6 3
57+
5
54+ 31+ 33+ 142
7 4 5 3
171
MC. Buoso et al. / Selenium in biological material
and control samples was also prepared. The quartz ampoules were sealed under clean conditions. The dry-to-wet mass ratio was determined for the materials used for irradiation. Two subsamples of approximately 5 g of each material were taken and dried in a temperature of 105°C for 2 h. Samples were irradiated with thermal neutrons in the reactor. The samples were placed in a two-stage sample holder, which was rotating during irradiation. The irradiation conditions were: 24 h at a thermal neutron flux of 8 x 1013 n/s. The cooling time was approximately 3 weeks. The y-activities of samples were measured using a HP-Ge well type detector. The following gamma lines were used: 1332 keV for 6oCo, 320 keV for ‘lCr, 1099 keV for 59Fe, 1077 keV for 86Rb, 401 keV for “Se, and 1115 keV for 65Zn. Recorded spectra were evaluated using the program GANAAS [4]. Quality control procedures were done with certified reference materials: NIST SRM 1572, apple leaves, cytrus leaves, NIST SRM 1515 and peach leaves NIST SRM 1547.
::I: 68 t
JO
F
4
B-
I
I
I
I
3. Results Altogether leaves from six trees in region USL 13 and ten trees in region USL 14 have been collected
I
/
I
I
I
126 t
I
I
i
i
Fig. 2. Se concentrations in Holm oak leaves sorted by district. District USL 13 had higher cancer mortability during the period 1980-1984 than district USL 14.
and analyzed. Here we present some representative results. X-ray spectra resulting from the irradiation of the leaves’ targets with 3.0 MeV proton beam contained peaks corresponding to characteristic X-rays of K, Ca, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, Br, Rb, Sr and Pb. Concentrations of these elements in the leaves’ targets were determined, and quality control has been achieved by analysis of certified reference material cytrus leaves (NIST SRM 1572). XRF results for Fe and Zn are in fair agreement with PIXE measurements. Table 1 shows 1.8 MeV PIXE and NAA results for samples collected in the district USL 14. Although not all samples were analyzed by both techniques, the agreement of the measured values is satisfactory. Data samples where both PIXE and NAA measurements were done are presented in fig. 1 in the form of a box and a whisker plot. The good agreement of PIXE and NAA is evident.
4. Conclusion Fig. 1. Multiple box- and whisker plot for NAA and PIXE data on the Se concentration in Holm oak leaves, for samples where both measurements were done.
When combined with appropriate preconcentration techniques, PIXE can be used for Se determination in biological material. The agreement with NAA meaIII. BIOMEDICAL SAMPLES
172
M.C. Buoso et al. / Selenium in biological material
surements is very good. In both cases the measurement of the Se concentration is a lengthy procedure. Additional useful information on the number of trace elements could be obtained in short measurements, with no sample pretreatment, using PIXE and XRF as analytical techniques. Although the results reported here should be considered as incomplete and preliminary, fig. 2 shows Se values in Holm oak tree leaves sorted by district. District USL 13 of Veneto Region in Italy which had a higher cancer mortality in the period 1980-1984, shows lower Se concentrations in oak leaves. This might be an indication of reduced Se bioavailability in this region, which results in a higher incidence of malignant disease.
The work along this line is in progress and one hopes to obtain more information needed for assessment of the selenium status in biological material.
References [l] V. Valkovic, Analysis of Biological Material for Trace Elements Using X-ray Spectroscopy (CRC Press, Boca Raton, Florida, 1980). [2] J.T. Salonen, G. Afithan, J.K. Huttesnen and P. Puska, Am. J. Epidemiol. 120 (1984) 588. [3] W. Maenhaut, L. De Reu and Vanderhaute, J. Nucl. Instr. and Meth. B3 (1984) 135. [4] GANAAS, Gamma spectrum Analysis, Activity Calculations and Neutron Activation Analysis (International Atomic Energy Agency, Vienna, 1991).
and Methods
in Physics Research
RIONil B
B75 (1993) 169-172
Beam Interactions with Materials&Atoms
Assessment of selenium status in biological material * M.C. Buoso ‘, S. Galassini a,b, M. Makarewicz ‘, F. Monti a,d, G. Moschini ‘TV,R. Ogris ‘, 0. Valkovic e and V. Valkovic Wa INFN, Laboratori Nazionali di Legnaro, Legnaro (Padova), Italy ’ Institute of Biological Chemistry, University of Verona, Italy ’ IAEA, Agency’s Laboratories, Seibersdorf; Austria d Physics Department, University of Padocla, Italy e Institut R. Boskovic, Zagreb, Croatia
Selenium is an essential trace element to man. Assessment of the selenium status, including its determination in biological materials, has been improved by the advances in analytical methodology. In this report we present the procedures used for selenium determination in different biological material by using nuclear analytical techniques, in particular PIXE, XRF and NAA. The difficulties associated with quality control of these measurements are discussed in some details. Examples of selenium determination in material are presented.
1. Introduction
There is clear, hard evidence that a geochemical environment can contribute to the maintenance of health in positive way [Il. Looking at the negative side, there is a sound evidence that a geochemical environment can be causally related to many diseases. Recently much attention has been paid to the group of elements called essential trace elements, one of them being selenium. Selenium occurs in nearly all materials of the Earth’s crust in variable concentrations rarely exceeding 0.05 ppm. Because of its variability in soil, the daily intake of selenium from food is variable and can range from 10 to 300 kg. Human activities are significantly impacting the global Se cycle, for example 1.5-2.5 times as much Se is mobilized through coal burning as by natural weather processes. LJpon combustion, coal-derived Se is preferentially partitioned to the submicrometer sized particles, which are easily transported over long distances. Selenium distribution in human tissues seems to reflect the existence of several pools for this element. Among the blood cells, platelets show a higher content of selenium than red blood cells. Selenium, when absorbed into blood, is translocated to various organs with a whole-body biological half-life of 34 d. Urine is the major route of excretion. Selenium levels in kidney, liver, spleen and heart are significantly correlated, indi* Work partially supported by the Commission pean Communities (Contract N. CI 1-0331-I(A)) Veneto (Contract N. 219/03/88).
of the Euroand Regione
0168-583X/93/$06.00
Publishers
0 1993 - El sevier Science
eating that for selenium no particular tissue (for instance the liver) is involved in selenium storage, but that selenium stores are distributed over the body. Some reports indicate a strong and significant correlation between hair selenium and liver selenium. When present in soluble forms Se is readily absorbed by plants, though differences between plant species are commonly observed. Several studies indicate that high levels of Se can inhibit cell proliferation and that the likely mechanism of action involved decreases protein synthesis. Most of these studies have demonstrated selenite to inhibit the growth and/or viability of tumor cells in culture. Recent studies have produced evidence that selenium may prevent cancer and stimulate the immune apparatus [2]. The main physiological role of this trace element is connected with its presence in various enzymes. Selenium is a constituent of enzyme glutathione peroxidase, protecting membranes from damage caused by the peroxidation of lipids. The aim of the present work is to demonstrate the use of nuclear analytical techniques in assessing the selenium status of the biological material. Although large number of selenium determination in blood, urine, tissue and different bioindicators have been performed, in this report we shall present results of measurement of Se and some other trace elements in oak tree leaves. The selection of oak free leaves as a Se exposure indicator was done because of i> the total volume of leaves being almost equal to the volume of roots and ii) the period of total leave replacement, corresponding to the period of Se integration, being three years.
B.V. All rights reserved
III. BIOMEDICAL
SAMPLES
170
MC. Buoso et al. / Selenium in biological material
2. Experimental
Leaves from oaks in two regions of Northern Italy (USL 13 and USL 141, one with increased cancer mortality, have been collected. The leaves were collected (1 kg from each tree) using surgical gloves, and were put in polyethylene bags. First of all, in order to remove dust particles, a blow of compressed air was used. Subsequently, the leaves were dried to constant weight in an oven at a temperature of about 100°C. Finally, clean, dry samples were ground in a blender, thoroughly mixed and then stored in new polyethylene bags. The analytical methods and elements determined are as follows, i) PIXE: Se (with preconcentration), K, Ca, Ti, Mn, Fe, Ni, Cu, Zn, Br, Rb, Sr, Pb; ii) XRF: Fe, Zn; iii) NAA: Se, Cr, Fe, Zn, Co. Proton induced X-ray emission spectroscopy measurements were performed with 1.8 MeV proton beam from AN 2000 Van de Graaff accelerator of the Laboratori Nazionali di Legnaro, Padova, Italy and with 3.0 MeV proton beam from EN tandem Van de Graaff accelerator of the Ruder Boskovic Institute, Zagreb, Croatia. Neutron activation analysis was done by sample irradiation with thermal neutrons in research reactor of Oesterreichische Forschungszentrum, Seibersdorf, Austria. X-ray fluorescence measurements were performed in the Laboratory for Nuclear Microanalysis, Ruder Boskovic Institute, Zagreb by using an X-ray tube excitation system with a MO anode. 2.1. PIXE measurements Samples for Se determination were prepared by the following procedures 131: 500 mg of leaf powder from each tree were placed in a 100 ml Pyrex flask with 13 ml of a mixture of 65% HNO, and 70% HCIO,, and heated to 120°C to evaporate perchloric acid 131.Then, 30% HCl was added until the HCi concentration reached 3M. After cooling, 600 pg of tellurium from a solution of KzTeO, (600 pgfml in 1N HCl) were added both as coprecipitating agent and as internal standard. Se and Te were then reduced to their elementary form by adding 4 ml of hydrazine dihydrochloride and 10 ml of HaSO,. After the selected reaction time (2 h), the precipitate was filtered using a suction Pyrex apparatus over a 0.4 km pore diameter Nucleopore. The diameter of the deposit was 10 mm. Accuracy of the measurements was verified by comparison with the Certified Standards Materials, NIST SRM 1571 Orchard Leaves (Se certified value: 0.08 +
0.01 mg/kg) and SRM 1568a Rice Flour (Se certified value: 0.4 rf:0.1 mg/kg). Samples for other trace element determinations were prepared from 1 g of fine leaf powder, without any pretreatment, as 5 mm thick pellets of 13 mm in diameter, using a stainless steel hand-press pellet maker. Samples were covered by aluminized Mylar in order to prevent charge buildup. In this case NIST SRM 1572, cytrus leaves, was used as standard reference material. The same samples were used for XRF measurements. 2.2. NAA measurements The samples were homogenized in the following way: Leaves were removed from the main rib and were grounded in a blender with a titanium knife. A fraction of the material was homogenized by the brittle fraction technique, using teflon boats cooled to the temperature of liquid nitrogen. Subsamples of about 0.2 g were weighted into quartz ampoules made from ultrapure quartz. A sufficient number of elemental standards
Table 1 Selenium concentration in Holm oak leaves in district USL 14 (w&z/kg) Sample code
NAA
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
55+ 47* 62f 74* 74* 91* 74* 14.5 + 43+ 70f 47+ 42+ 31+ 23+ 30* 24+ 47+ 41+
19 20 21 22 23 24 25 26 27
20* 215 17* 39*
PIXE 4 4 4 5 5 5 5 10 4 3 3 4 2 1 2 2 2 2 2 2 1 2
57+
64+
6
8
91% 5 1so+ 57+ 65& 70+ 62t 34*
20 5 7 7 5 7
21*
4
34+
6
34+ 22+
6 3
57+
5
54+ 31+ 33+ 142
7 4 5 3
171
MC. Buoso et al. / Selenium in biological material
and control samples was also prepared. The quartz ampoules were sealed under clean conditions. The dry-to-wet mass ratio was determined for the materials used for irradiation. Two subsamples of approximately 5 g of each material were taken and dried in a temperature of 105°C for 2 h. Samples were irradiated with thermal neutrons in the reactor. The samples were placed in a two-stage sample holder, which was rotating during irradiation. The irradiation conditions were: 24 h at a thermal neutron flux of 8 x 1013 n/s. The cooling time was approximately 3 weeks. The y-activities of samples were measured using a HP-Ge well type detector. The following gamma lines were used: 1332 keV for 6oCo, 320 keV for ‘lCr, 1099 keV for 59Fe, 1077 keV for 86Rb, 401 keV for “Se, and 1115 keV for 65Zn. Recorded spectra were evaluated using the program GANAAS [4]. Quality control procedures were done with certified reference materials: NIST SRM 1572, apple leaves, cytrus leaves, NIST SRM 1515 and peach leaves NIST SRM 1547.
::I: 68 t
JO
F
4
B-
I
I
I
I
3. Results Altogether leaves from six trees in region USL 13 and ten trees in region USL 14 have been collected
I
/
I
I
I
126 t
I
I
i
i
Fig. 2. Se concentrations in Holm oak leaves sorted by district. District USL 13 had higher cancer mortability during the period 1980-1984 than district USL 14.
and analyzed. Here we present some representative results. X-ray spectra resulting from the irradiation of the leaves’ targets with 3.0 MeV proton beam contained peaks corresponding to characteristic X-rays of K, Ca, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, Br, Rb, Sr and Pb. Concentrations of these elements in the leaves’ targets were determined, and quality control has been achieved by analysis of certified reference material cytrus leaves (NIST SRM 1572). XRF results for Fe and Zn are in fair agreement with PIXE measurements. Table 1 shows 1.8 MeV PIXE and NAA results for samples collected in the district USL 14. Although not all samples were analyzed by both techniques, the agreement of the measured values is satisfactory. Data samples where both PIXE and NAA measurements were done are presented in fig. 1 in the form of a box and a whisker plot. The good agreement of PIXE and NAA is evident.
4. Conclusion Fig. 1. Multiple box- and whisker plot for NAA and PIXE data on the Se concentration in Holm oak leaves, for samples where both measurements were done.
When combined with appropriate preconcentration techniques, PIXE can be used for Se determination in biological material. The agreement with NAA meaIII. BIOMEDICAL SAMPLES
172
M.C. Buoso et al. / Selenium in biological material
surements is very good. In both cases the measurement of the Se concentration is a lengthy procedure. Additional useful information on the number of trace elements could be obtained in short measurements, with no sample pretreatment, using PIXE and XRF as analytical techniques. Although the results reported here should be considered as incomplete and preliminary, fig. 2 shows Se values in Holm oak tree leaves sorted by district. District USL 13 of Veneto Region in Italy which had a higher cancer mortality in the period 1980-1984, shows lower Se concentrations in oak leaves. This might be an indication of reduced Se bioavailability in this region, which results in a higher incidence of malignant disease.
The work along this line is in progress and one hopes to obtain more information needed for assessment of the selenium status in biological material.
References [l] V. Valkovic, Analysis of Biological Material for Trace Elements Using X-ray Spectroscopy (CRC Press, Boca Raton, Florida, 1980). [2] J.T. Salonen, G. Afithan, J.K. Huttesnen and P. Puska, Am. J. Epidemiol. 120 (1984) 588. [3] W. Maenhaut, L. De Reu and Vanderhaute, J. Nucl. Instr. and Meth. B3 (1984) 135. [4] GANAAS, Gamma spectrum Analysis, Activity Calculations and Neutron Activation Analysis (International Atomic Energy Agency, Vienna, 1991).